The overall goal of the research proposed in this renewal application is to understand how HIV-1 Gag is trafficked through the cell, targeted to membranes and assembled into virions. During the previous granting period, we established that HIV-1 assembly is an ordered process that proceeds through sequential intermediates. We showed that Gag is localized to the plasma membrane and that targeting of Gag to membrane raft-like domains, "barges" promotes efficient particle formation. In addition, we identified a motif (IL) within Gag that regulates its association with multivesicular bodies (MVBs). In the next renewal period, we plan to extend these studies by elucidating the mechanisms for Gag trafficking, subcellular localization, and interaction with the cellular endocytic and exocytic pathways. The proposed research is designed to: 1. Determine the mechanisms that regulate Gag targeting to the plasma membrane vs MVBs in different cell types. We will test the following hypotheses: A) MVB formation is necessaryfor Gag mediated assembly; B) Rab27a regulates exocytosis of Gag-containing MVBs and/or secretory lysosomes. C) Ca++ flux regulates exocytosis of Gag-containing MVBs. In addition, D) We will use TIR-FM (total internal reflection fluorescence microscopy) to visualize and quantitate Gag mediated exocytic events occurring within 100 nm of the plasma membrane. 2. Determine whether Gag expression alters the flux of cellular proteins through the MVB pathway. We will determine whether Gag alters the functioning of the cellular multivesicular body pathway by examining the kinetics of EGF Receptor downregulation and signaling in Gag transfected cells, in pHXB2 transfected cells, and in HIV-1 infected cells. In addition, we will determine whether Gag alters downregulation and signaling via the chemokine and HIV-1 co-receptor, CXCR4, in T cells. 3. Define the mechanisms of Gag membrane targeting, trafficking and assembly by imaging in live and fixed cells. Inducible methods for Gag expression will be exploited to perform time lapse imaging of intracellular trafficking in real time. Multicolor imaging with site-specific fluorescein derivatives (FLASH and ReAsH) will be used to determine the order in which Gag assembly sites are formed and maintained in the cell.